Structure of the bc1-cbb3 respiratory supercomplex from Pseudomonas aeruginosa.

Energy conversion by electron transport chains occurs through the sequential transfer of electrons between protein complexes and intermediate electron carriers, creating the proton motive force that enables ATP synthesis and membrane transport. These protein complexes can also form higher order assemblies known as respiratory supercomplexes (SCs). The electron transport chain of the opportunistic pathogen Pseudomonas aeruginosa is closely linked with its ability to invade host tissue, tolerate harsh conditions, and resist antibiotics but is poorly characterized. Here, we determine the structure of a P. aeruginosa SC that forms between the quinol:cytochrome c oxidoreductase (cytochrome bc1) and one of the organism's terminal oxidases, cytochrome cbb3, which is found only in some bacteria. Remarkably, the SC structure also includes two intermediate electron carriers: a diheme cytochrome c4 and a single heme cytochrome c5. Together, these proteins allow electron transfer from ubiquinol in cytochrome bc1 to oxygen in cytochrome cbb3. We also present evidence that different isoforms of cytochrome cbb3 can participate in formation of this SC without changing the overall SC architecture. Incorporating these different subunit isoforms into the SC would allow the bacterium to adapt to different environmental conditions. Bioinformatic analysis focusing on structural motifs in the SC suggests that cytochrome bc1-cbb3 SCs also exist in other bacterial pathogens.

Impact of Aneuploidy and Chromosome 9p Loss on Tumor Immune Microenvironment and Immune Checkpoint Inhibitor Efficacy in NSCLC.

INTRODUCTION: Although gene-level copy number alterations have been studied as a potential biomarker of immunotherapy efficacy in NSCLC, the impact of aneuploidy burden and chromosomal arm-level events on immune checkpoint inhibitor (ICI) efficacy in NSCLC is uncertain. METHODS: Patients who received programmed cell death protein 1 or programmed death-ligand 1 (PD-L1) inhibitor at two academic centers were included. Across all 22 chromosomes analyzed, an arm was considered altered if at least 70% of its territory was either gained or deleted. Among nonsquamous NSCLCs which underwent targeted next-generation sequencing, we retrospectively quantified aneuploidy using the adjusted fraction of chromosomal arm alterations (FAA), defined as the number of altered chromosome arms divided by the number of chromosome arms assessed, adjusted for tumor purity. RESULTS: Among 2293 nonsquamous NSCLCs identified, the median FAA increased with more advanced cancer stage and decreased with higher PD-L1 tumor proportion score (TPS) levels (median FAA in TPS < 1%: 0.09, TPS 1%-49%: 0.08, TPS ≥ 50%: 0.05, p < 0.0001). There was a very weak correlation between FAA and tumor mutational burden when taken as continuous variables (R: 0.07, p = 0.0005). A total of 765 advanced nonsquamous NSCLCs with available FAA values were treated with ICIs. With decreasing FAA tertiles, there was a progressive improvement in objective response rate (ORR 15.1% in upper tertile versus 23.2% in middle tertile versus 28.4% in lowest tertile, p = 0.001), median progression-free survival (mPFS 2.5 versus 3.3 versus 4.1 mo, p < 0.0001), and median overall survival (mOS 12.5 versus 13.9 versus 16.4 mo, p = 0.006), respectively. In the arm-level enrichment analysis, chromosome 9p loss (OR = 0.22, Q = 0.0002) and chromosome 1q gain (OR = 0.43, Q = 0.002) were significantly enriched in ICI nonresponders after false discovery rate adjustment. Compared with NSCLCs without chromosome 9p loss (n = 452), those with 9p loss (n = 154) had a lower ORR (28.1% versus 7.8%, p < 0.0001), a shorter mPFS (4.1 versus 2.3 mo, p < 0.0001), and a shorter mOS (18.0 versus 9.6 mo, p < 0.0001) to immunotherapy. In addition, among NSCLCs with high PD-L1 expression (TPS ≥ 50%), chromosome 9p loss was associated with lower ORR (43% versus 6%, p < 0.0001), shorter mPFS (6.4 versus 2.6 mo, p = 0.0006), and shorter mOS (30.2 versus 14.3 mo, p = 0.0008) to immunotherapy compared with NSCLCs without 9p loss. In multivariable analysis, adjusting for key variables including FAA, chromosome 9p loss, but not 1q gain, retained a significant impact on ORR (hazard ratio [HR] = 0.25, p < 0.001), mPFS (HR = 1.49, p = 0.001), and mOS (HR = 1.47, p = 0.003). Multiplexed immunofluorescence and computational deconvolution of RNA sequencing data revealed that tumors with either high FAA levels or chromosome 9p loss had significantly fewer tumor-associated cytotoxic immune cells. CONCLUSIONS: Nonsquamous NSCLCs with high aneuploidy and chromosome 9p loss have a distinct tumor immune microenvironment and less favorable outcomes to ICIs.

Brief Communication on Pathologic Assessment of Persistent Stable Metastatic Lesions in Patients Treated With Anti-CTLA-4 or Anti-CTLA-4 + Anti-PD-1 Therapy.

Despite the wide use of immune checkpoint inhibition for the treatment of melanoma, the mechanisms leading to long-term stable disease are incompletely understood. Patients with metastatic melanoma who had received ipilimumab alone or ipilimumab plus nivolumab 2+years prior and attained at least 6 months of stable disease were identified. Positron emission tomography/computed tomography (PET/CT) was performed. Pretreatment and posttreatment biopsies of areas of stable disease were assessed for tumor, fibrosis, and inflammation. Seven patients underwent PET/CT and tissue biopsy. Fluorodeoxyglucose avid lesions on PET/CT ranged from no activity to an SUV of 22. In 6 patients, the residual stable lesions were composed of necrosis and fibrosis with a prominent pigment containing macrophages and no residual melanoma. In 1 patient, a nodal lesion demonstrated melanoma with active inflammation. In most patients with durable stable disease after treatment with ipilimumab or ipilimumab/nivolumab, residual lesions demonstrated predominantly necrosis and fibrosis consistent with resolving lesions. The presence of melanophages in these samples may suggest ongoing immune surveillance. One patient did demonstrate residual melanoma, indicating the need for ongoing monitoring of this patient population.

Lysosomal swelling and lysis mediate delivery of C3 toxins to their cytoplasmic targets.

Unlike other cholera-like toxins that contain separate binding/translocation and catalytic subunits, C3-like mono-ADP-ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently labeled C3larvinA. Following binding to the macrophage membrane in a discontinuous punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA-treated cells underwent massive swelling over the course of 1-4 h. Lysosomal swelling preceded the extensive rearrangement of the cellular F-actin caused by ADP-ribosylation of cytosolic Rho-GTPases. This suggested that lysosome swelling might be required for the escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F-actin rearrangement. Toxin-induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano-sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodeling activities of C3larvinA with that of two related toxins, C3larvintrunc and Plx2A, highlighted the importance of the N-terminal α1 -helix for lysosomal swelling and successful intoxication.

Inference of single cell profiles from histology stains with the Single-Cell omics from Histology Analysis Framework (SCHAF).

Tissue biology involves an intricate balance between cell-intrinsic processes and interactions between cells organized in specific spatial patterns, which can be respectively captured by single-cell profiling methods, such as single-cell RNA-seq (scRNA-seq), and histology imaging data, such as Hematoxylin-and-Eosin (H&E) stains. While single-cell profiles provide rich molecular information, they can be challenging to collect routinely and do not have spatial resolution. Conversely, histological H&E assays have been a cornerstone of tissue pathology for decades, but do not directly report on molecular details, although the observed structure they capture arises from molecules and cells. Here, we leverage adversarial machine learning to develop SCHAF (Single-Cell omics from Histology Analysis Framework), to generate a tissue sample's spatially-resolved single-cell omics dataset from its H&E histology image. We demonstrate SCHAF on two types of human tumors-from lung and metastatic breast cancer-training with matched samples analyzed by both sc/snRNA-seq and by H&E staining. SCHAF generated appropriate single-cell profiles from histology images in test data, related them spatially, and compared well to ground-truth scRNA-Seq, expert pathologist annotations, or direct MERFISH measurements. SCHAF opens the way to next-generation H&E2.0 analyses and an integrated understanding of cell and tissue biology in health and disease.

Correction: Comprehensive Immunoprofiling of High-risk Oral Proliferative and Localized Leukoplakia.

[This corrects the article DOI: 10.1158/2767-9764.CRC-21-0060.][This corrects the article DOI: 10.1158/2767-9764.CRC-21-0060.].

A Structural Approach to Anti-Virulence: A Discovery Pipeline.

The anti-virulence strategy is designed to prevent bacterial virulence factors produced by pathogenic bacteria from initiating and sustaining an infection. One family of bacterial virulence factors is the mono-ADP-ribosyltransferase toxins, which are produced by pathogens as tools to compromise the target host cell. These toxins are bacterial enzymes that exploit host cellular NAD+ as the donor substrate to modify an essential macromolecule acceptor target in the host cell. This biochemical reaction modifies the target macromolecule (often protein or DNA) and functions in a binary fashion to turn the target activity on or off by blocking or impairing a critical process or pathway in the host. A structural biology approach to the anti-virulence method to neutralize the cytotoxic effect of these factors requires the search and design of small molecules that bind tightly to the enzyme active site and prevent catalytic function essentially disarming the pathogen. This method requires a high-resolution structure to serve as the model for small molecule inhibitor development, which illuminates the path to drug development. This alternative strategy to antibiotic therapy represents a paradigm shift that may circumvent multi-drug resistance in the offending microbe through anti-virulence therapy. In this report, the rationale for the anti-virulence structural approach will be discussed along with recent efforts to apply this method to treat honey bee diseases using natural products.

Low peripheral blood derived neutrophil-to-lymphocyte ratio (dNLR) is associated with increased tumor T-cell infiltration and favorable outcomes to first-line pembrolizumab in non-small cell lung cancer.

BACKGROUND: An elevated peripheral blood derived neutrophil-to-lymphocyte ratio (dNLR) is a negative prognostic marker for patients with non-small cell lung cancer (NSCLC) receiving chemotherapy and immune checkpoint inhibitors. Whether dNLR is also associated with clinical outcomes to first-line pembrolizumab among patients with NSCLC and a programmed cell death ligand 1 (PD-L1) Tumor Proportion Score (TPS) of ≥50% is uncertain. How dNLR relates to the tumor immune microenvironment is also unclear. METHODS: In two participating academic centers, we retrospectively analyzed the dNLR (defined as the absolute neutrophil count/white cell count - absolute neutrophil count) prior to initiation of first-line pembrolizumab in patients with metastatic NSCLC and a PD-L1 TPS ≥50% and lacking genomic alterations in EGFR and ALK. An unbiased recursive partitioning algorithm was used to investigate an optimal dNLR cut-off with respect to objective response rate (ORR). Multiplexed immunofluorescence for CD8+, FOXP3+, PD-1+, and PD-L1 was performed on a separate cohort of NSCLCs to determine the immunophenotype associated with dNLR. RESULTS: A total of 221 patients treated with first-line pembrolizumab were included in this study. The optimal dNLR cut-off to differentiate treatment responders from non-responders was 2.6. Compared with patients with a dNLR ≥2.6 (n=97), patients with dNLR <2.6 (n=124) had a significantly higher ORR (52.4% vs 24.7%, p<0.001), a significantly longer median progression-free survival (mPFS 10.4 vs 3.4 months, HR 0.48, 95% CI 0.35 to 0.66, p<0.001), and a significantly longer median overall survival (mOS 36.6 vs 9.8 months, HR 0.34, 95% CI 0.23 to 0.49, p<0.001). After adjusting for age, sex, tobacco use, performance status, histology, serum albumin level, oncogenic driver status, and PD-L1 distribution (50%-89% vs ≥90%), a dNLR <2.6 was confirmed to be an independent predictor of longer mPFS (HR 0.47, 95% CI 0.33 to 0.67, p<0.001) and mOS (HR 0.32, 95% CI 0.21 to 0.49, p<0.001). Among advanced NSCLC samples with a PD-L1 TPS of ≥50%, those with a dNLR <2.6 had significantly higher numbers of tumor-associated CD8+, FOXP3+, PD-1 +immune cells, and PD-1 +CD8+T cells than those with a dNLR ≥2.6. CONCLUSIONS: Among patients with NSCLC and a PD-L1 TPS ≥50%, a low dNLR has a distinct immune tumor microenvironment and more favorable outcomes to first-line pembrolizumab.

Anti-Virulence Strategy against the Honey Bee Pathogenic Bacterium Paenibacillus larvae via Small Molecule Inhibitors of the Bacterial Toxin Plx2A.

American Foulbrood, caused by Paenibacillus larvae, is the most devastating bacterial honey bee brood disease. Finding a treatment against American Foulbrood would be a huge breakthrough in the battle against the disease. Recently, small molecule inhibitors against virulence factors have been suggested as candidates for the development of anti-virulence strategies against bacterial infections. We therefore screened an in-house library of synthetic small molecules and a library of flavonoid natural products, identifying the synthetic compound M3 and two natural, plant-derived small molecules, Acacetin and Baicalein, as putative inhibitors of the recently identified P. larvae toxin Plx2A. All three inhibitors were potent in in vitro enzyme activity assays and two compounds were shown to protect insect cells against Plx2A intoxication. However, when tested in exposure bioassays with honey bee larvae, no effect on mortality could be observed for the synthetic or the plant-derived inhibitors, thus suggesting that the pathogenesis strategies of P. larvae are likely to be too complex to be disarmed in an anti-virulence strategy aimed at a single virulence factor. Our study also underscores the importance of not only testing substances in in vitro or cell culture assays, but also testing the compounds in P. larvae-infected honey bee larvae.

Comprehensive Immunoprofiling of High-Risk Oral Proliferative and Localized Leukoplakia.

Oral leukoplakia is common and may, in some cases, progress to carcinoma. Proliferative leukoplakia is a progressive, often multifocal subtype with a high rate of malignant transformation compared with the more common localized leukoplakia. We hypothesized that the immune microenvironment and gene expression patterns would be distinct for proliferative leukoplakia compared with localized leukoplakia. We summarize key clinicopathologic features among proliferative leukoplakia and localized leukoplakia and compare cancer-free survival (CFS) between subgroups. We analyze immunologic gene expression profiling in proliferative leukoplakia and localized leukoplakia tissue samples (NanoString PanCancer Immune Oncology Profiling). We integrate immune cell activation and spatial distribution patterns in tissue samples using multiplexed immunofluorescence and digital image capture to further define proliferative leukoplakia and localized leukoplakia. Among N = 58 patients (proliferative leukoplakia, n = 29; localized leukoplakia, n = 29), only the clinical diagnosis of proliferative leukoplakia was associated with significantly decreased CFS (HR, 11.25; P < 0.01; 5-year CFS 46.8% and 83.6% among patients with proliferative leukoplakia and localized leukoplakia, respectively). CD8+ T cells and T regulatory (Treg) were more abundant among proliferative leukoplakia samples (P < 0.01) regardless of degree of epithelial dysplasia, and often colocalized to the dysplasia-stromal interface. Gene set analysis identified granzyme M as the most differentially expressed gene favoring the proliferative leukoplakia subgroup (log2 fold change, 1.93; P adj < 0.001). Programmed death ligand 1 (PD-L1) was comparatively overexpressed among proliferative leukoplakia samples, with higher (>5) PD-L1 scores predicting worse CFS (P adj < 0.01). Proliferative leukoplakia predicts a high rate of malignant transformation within 5 years of diagnosis. A prominent CD8+ T-cell and Treg signature along with relative PD-L1 overexpression compared with localized leukoplakia provides strong rationale for PD-1/PD-L1 axis blockade using preventative immunotherapy. Significance: This is the first in-depth profiling effort to immunologically characterize high-risk proliferative leukoplakia as compared with the more common localized leukoplakia. We observed a notable cytotoxic T-cell and Treg signature with relative overexpression of PD-L1 in high-risk proliferative leukoplakia providing a strong preclinical rationale for investigating PD-1/PD-L1 axis blockade in this disease as preventative immunotherapy.

The N-terminus of Paenibacillus larvae C3larvinA modulates catalytic efficiency.

C3larvinA was recently described as a mono-ADP-ribosyltransferase (mART) toxin from the enterobacterial repetitive intergenic consensus (ERIC) III genotype of the agricultural pathogen, Paenibacillus larvae. It was shown to be the full-length, functional version of the previously described C3larvintrunc toxin, due to a 33-residue extension of the N-terminus of the protein. In the present study, a series of deletions and substitutions were made to the N-terminus of C3larvinA to assess the contribution of the α1-helix to toxin structure and function. Catalytic characterization of these variants identified Asp23 and Ala31 residues as supportive to enzymatic function. A third residue, Lys36, was also found to contribute to the catalytic activity of the enzyme. Analysis of the C3larvinA homology model revealed that these three residues were participating in a series of interactions to properly orient both the Q-X-E and S-T-S motifs. Ala31 and Lys36 were found to associate with a structural network of residues previously identified in silico, whereas Asp23 forms novel interactions not previously described. At last, the membrane translocation activity into host target cells of each variant was assessed, highlighting a possible relationship between protein dipole and target cell entry.

Characterization of C3larvinA, a novel RhoA-targeting ADP-ribosyltransferase toxin produced by the honey bee pathogen, Paenibacillus larvae.

C3larvinA is a putative virulence factor produced by Paenibacillus larvae enterobacterial-repetitive-intergenic-consensus (ERIC) III/IV (strain 11-8051). Biochemical, functional and structural analyses of C3larvinA revealed that it belongs to the C3-like mono-ADP-ribosylating toxin subgroup. Mammalian RhoA was the target substrate for its transferase activity suggesting that it may be the biological target of C3larvinA. The kinetic parameters of the NAD+ substrate for the transferase (KM = 75 ± 10 µM) and glycohydrolase (GH) (KM = 107 ± 20 µM) reactions were typical for a C3-like bacterial toxin, including the Plx2A virulence factor from Paenibacillus larvae ERIC I. Upon cytoplasmic expression in yeast, C3larvinA caused a growth-defective phenotype indicating that it is an active C3-like toxin and is cytotoxic to eukaryotic cells. The catalytic variant of the Q187-X-E189 motif in C3larvinA showed no cytotoxicity toward yeast confirming that the cytotoxicity of this factor depends on its enzymatic activity. A homology consensus model of C3larvinA with NAD+ substrate was built on the structure of Plx2A, provided additional confirmation that C3larvinA is a member of the C3-like mono-ADP-ribosylating toxin subgroup. A homology model of C3larvinA with NADH and RhoA was built on the structure of the C3cer-NADH-RhoA complex which provided further evidence that C3larvinA is a C3-like toxin that shares an identical catalytic mechanism with C3cer from Bacillus cereus. C3larvinA induced actin cytoskeleton reorganization in murine macrophages, whereas in insect cells, vacuolization and bi-nucleated cells were observed. These cellular effects are consistent with C3larvinA disrupting RhoA function by covalent modification that is shared among C3-like bacterial toxins.

Pressure-induced structural modifications of rare-earth hafnate pyrochlore.

Complex oxides with the pyrochlore (A2B2O7) and defect-fluorite ((A,B)4O7) structure-types undergo structural transformations under high-pressure. Rare-earth hafnates (A2Hf2O7) form the pyrochlore structure for A = La-Tb and the defect-fluorite structure for A = Dy-Lu. High-pressure transformations in A2Hf2O7 pyrochlore (A = Sm, Eu, Gd) and defect-fluorite (A = Dy, Y, Yb) were investigated up to ~50 GPa and characterized by in situ Raman spectroscopy and synchrotron x-ray diffraction (XRD). Raman spectra at ambient pressure revealed that all compositions, including the defect-fluorites, have some pyrochlore-type short-range order. In situ high-pressure synchrotron XRD showed that all of the rare earth hafnates investigated undergo a pressure-induced phase transition to a cotunnite-like (orthorhombic) structure that begins between 18 and 25 GPa. The phase transition to the cotunnite-like structure is not complete at 50 GPa, and upon release of pressure, the hafnates transform to defect-fluorite with an amorphous component. For all compositions, in situ Raman spectroscopy showed that disordering occurs gradually with increasing pressure. Pyrochlore-structured hafnates retain their short-range order to a higher pressure (30 GPa vs. <10 GPa) than defect-fluorite-structured hafnates. Rare earth hafnates quenched from 50 GPa show Raman spectra consistent with weberite-type structures, as also reported for irradiated rare-earth stannates. The second-order Birch-Murnaghan equation of state fit gives a bulk modulus of ~250 GPa for hafnates with the pyrochlore structure, and ~400 GPa for hafnates with the defect-fluorite structure. Dy2Hf2O7 is intermediate in its response, with some pyrochlore-type ordering, based on Raman spectroscopy and the equation of state, with a bulk modulus of ~300 GPa. As predicted based on the similar ionic radius of Zr4+ and Hf4+, rare-earth hafnates show similar behavior to that reported for rare earth zirconates at high pressure.